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J. Biol. Chem., Vol. 276, Issue 37, 34530-34536, September 14, 2001

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Glucose-induced Oscillations in Cytoplasmic Free Ca²⁺ Concentration Precede Oscillations in Mitochondrial Membrane Potential in the Pancreatic -Cell^*

Henrik Kindmark^§, Martin Köhler, Graham Brown, Robert Bränström, Olof Larsson, and Per-Olof Berggren

From the Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden

Received for publication, March 20, 2001, and in revised form, June 18, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Using dual excitation and fixed emission fluorescence microscopy, we were able to measure changes in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) and mitochondrial membrane potential simultaneously in the pancreatic -cell. The -cells were exposed to a combination of the Ca²⁺ indicator fura-2/AM and the indicator of mitochondrial membrane potential, rhodamine 123 (Rh123). Using simultaneous measurements of mitochondrial membrane potential and [Ca²⁺]_i during glucose stimulation, it was possible to measure the time lag between the onset of mitochondrial hyperpolarization and changes in [Ca²⁺]_i. Glucose-induced oscillations in [Ca²⁺]_i were followed by transient depolarizations of mitochondrial membrane potential. These results are compatible with a model in which nadirs in [Ca²⁺]_i oscillations are generated by a transient, Ca²⁺-induced inhibition of mitochondrial metabolism resulting in a temporary fall in the cytoplasmic ATP/ADP ratio, opening of plasma membrane K_ATP channels, repolarization of the plasma membrane, and thus transient closure of voltage-gated L-type Ca²⁺ channels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glucose stimulates insulin secretion as a consequence of being metabolized by the pancreatic -cell. Metabolism of glucose leads to an increase in the cytosolic ATP/ADP ratio, closure of ATP-dependent K⁺ channels (K_ATP channels)¹ in the plasma membrane, and thus depolarization of the -cell (1, 2). This results in Ca²⁺ influx through voltage-gated L-type Ca²⁺ channels, an increase in cytoplasmic free Ca²⁺ concentration, [Ca²⁺]_i, and thereby release of insulin. [Ca²⁺]_i does not increase to a sustained elevated level. On the contrary, stimulation of pancreatic -cells with intermediate concentrations of glucose results in oscillations in electrical activity and [Ca²⁺]_i (3-5). The detailed regulation of these oscillations at the single cell level is not clear. In a previous study we reported evidence suggesting that K_ATP channel activity oscillates in parallel with glucose-induced [Ca²⁺]_i oscillations (5).

Previous studies have measured [Ca²⁺]_i in pancreatic -cells using the dye fura-2 (4, 6, 7). Measurements of mitochondrial membrane potential in these cells, using the lipophilic cationic dye rhodamine 123 (Rh123), have also been reported (8). Simultaneous measurements of [Ca²⁺]_i and mitochondrial membrane potential in the pancreatic -cell would be of great interest and could provide important information about the possible correlation between mitochondrial responses to glucose stimulation and events at the plasma membrane of the -cell. Such simultaneous measurements have recently been performed in neurons (9, 10) and in mouse -cells (11). To investigate the relationship between mitochondrial membrane potential and [Ca²⁺]_i in the -cell, we loaded -cells with fura-2 and Rh123 and applied the method of simultaneous measurements of these two parameters. Our methodological evaluation included an investigation of cellular Rh123 distribution as observed with confocal microscopy. We also estimated the contribution to emitted light from both dyes at the respective excitation wavelength, allowing us to correct for contaminating fluorescence.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

All reagents were of analytical grade and Millipore water was used. Bovine serum albumin fraction V was from Sigma, collagenase was from Roche Molecular Biochemicals, and fura-2/AM as well as Rh123 were from Molecular Probes Europe BV (Leiden, The Netherlands). Statistical summaries of data are expressed as the mean ± S.E. Where appropriate, the possible statistical significance of differences between two sets of data was tested using Student's t test for unpaired data. In all experimental protocols, -cells from at least three different animals were used, if not otherwise specified.

Animals and Preparation of Cells-- Adult obese mice (gene symbol ob/ob) of both sexes were obtained from a local colony and starved for 24 h. The animals were killed by decapitation and the islets isolated using a collagenase technique (12). The islets of these mice contain more than 90% -cells (13). A cell suspension was prepared and washed essentially as described previously (14). The cells were suspended in RPMI 1640 culture medium (Flow Laboratories) containing 11 mM glucose supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin (Life Technologies, Inc.). The cell suspension was seeded onto coverslips, and the cells allowed to attach for 2 h and then cultured for up to 3 days in the above medium.

Media-- The basal medium used for preparation of cells as well as experiments was a HEPES buffer, pH 7.4, with Cl as the sole anion (15), containing 3 mM glucose, 1.28 mM Ca²⁺, and 0.1% bovine serum albumin.

Measurements of [Ca²⁺]_i Simultaneously with Mitochondrial Membrane Potential-- Prior to simultaneous measurements of [Ca²⁺]_i and mitochondrial membrane potential, -cells attached to coverslips in basal medium were incubated in 2 or 3 µM fura-2/AM for 30 min at 37 °C in the presence of 0.1% bovine serum albumin. 2, 8, 12, 16, or 32 µM Rh123 was added to the incubation buffer during the last 10 min of fura-2 loading, and the coverslips were then washed. In studies of hepatocytes, the concentration of Rh123 reaches equilibrium across the plasma membrane after incubation of the cells for 10 min in a buffer containing the probe (16). Measurements were carried out with a SPEX fluorolog-2 CM1T11I system connected to an inverted microscope (Zeiss, Axiovert 35 M). The monochromators on the excitation side were set at 380 nm (fura-2) and 500 nm (Rh123). On the emission side, a band-pass filter (515-565 nm) was used. Cells were maintained at 37 °C by temperature-controlled metal jackets on both the perfusion chamber and the objective lens.

Measurements and Analysis of NAD(P)H and Fura-2 Fluorescence-- -Cells attached to coverslips were illuminated with light at 350 nm (380 nm for Fura-2), obtained using a band-pass filter in a system including a Leica DMIRB microscope with a PL Fluotar 40×/1.00-0.50 oil objective. Emitted light was collected through a 400-510 nm (500-530 nm for fura-2) band-pass filter, detected using a LSR Astrocam camera with CCD-type Site 502AB-1, and analyzed with LSR Merlin software (PerkinElmer Life Sciences, Cambridge, UK). Average fluorescence from separate cells was monitored and analyzed with spectral analysis to reveal the presence of different frequencies. The power spectral density function in MATLAB (The MathWorks Inc., Natick, MA) was used with Welch's method and a Kaiser window.

Confocal Microscopy Experiments-- Pancreatic -cells on glass coverslips were incubated with 3 µM fura-2/AM for 30 min and, during the last 10 min, also with various concentrations of Rh123. The -cells were then washed. The coverslips formed the bottom of a perfusion chamber and were kept at 37 °C by temperature-controlled metal jackets on both the perfusion chamber and the objective lens. Fluorescence was recorded using a confocal laser scanning microscope system (CLSM, Leica Lasertechnik GmbH, Heidelberg, Germany). Rhodamine 123 was excited with the 488 nm line of a krypton-argon laser (Omnichrome, Chino, CA), and emitted light was detected using a long-pass 515 nm barrier filter. The pinhole was set to give a confocal section thickness of no greater than 1 µm, using a 100× Leitz PL Fluotar oil immersion objective, NA 1.32. Quantification of Rh123 fluorescence from the nucleus and the cytoplasm was made with the objective focused on the center of the nucleus. This ensured minimal interference between nuclear and cytoplasmic areas and also guaranteed that fluorescence from the nuclear area was indeed originating from the nucleus. Images were processed using ImageTool (University of Texas Health Science Center), by which fluorescence intensities were averaged within defined regions of the nucleus and cytoplasm.

Patch Clamp Experiments-- The perforated whole-cell configuration of the patch clamp technique (17, 18) was used. Pipettes were pulled from borosilicate glass coated with Sylgard resin (Dow Corning) near the tips, fire-polished, and having a resistance between 4 and 8 megaohms. Current and voltage were recorded using an Axopatch 200 patch clamp amplifier (Axon Instruments, Inc, Foster City, CA). During the experiments, the current and voltage signals were stored using a VR-100A digital recorder (Instrutech Corp.) and a high resolution video cassette recorder (JVC). The pipette solution consisted of (in mM): 10 KCl, 76 K₂SO₄, 10 NaCl, 1 MgCl₂, 10 HEPES-NaOH, pH 7.35, and 200 µg/ml amphotericin B (dissolved in Me₂SO; final concentration of Me₂SO was less than 0.1%). The extracellular solution consisted of the HEPES buffer described above under "Media."

For the experiments employing voltage clamp in combination with measurement of Rh123 fluorescence in Rh123-loaded -cells, a HEKA EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht, Germany) was used in conjunction with a Concord photometer and digital imaging setup (PerkinElmer Life Sciences, Cambridge, UK). 488 nm was used as the excitation wavelength in these experiments, and emitted light was collected through a band-pass filter (515-565 nm). All data acquisition was done through the Concord instrument, which thus also registered voltage pulses versus time.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Contribution by Fura-2 and Rh123 to Fluorescence at 515-565 nm using 380 and 500 nm Excitation-- Excitation at 380 nm is intended to cause fura-2 to fluoresce, whereas 500 nm is intended to excite Rh123. When both fluorescent dyes are used simultaneously, it is important to determine whether emitted fluorescence at either excitation wavelength is produced exclusively by the expected dye or whether there is contaminating fluorescence from the other dye. According to our measurements, the fluorescence from fura-2-loaded cells at 500 nm excitation (F) is the same as background fluorescence from unloaded cells (4990 ± 257 cps versus 4900 ± 920 cps for small aggregates consisting of 4-8 cells and 1920 ± 113 cps versus 2150 ± 420 cps for single cells; n = 10 or more using -cells from 5 mice for each of these protocols). Therefore we can consider all fluorescence changes from 500 nm excitation to originate from Rh123. Cells loaded with only Rh123 demonstrated the strongest fluorescence at 500 nm excitation (F), whereas excitation at 380 nm gave only a minor fluorescence (F). F was linearly dependent on F, and thus we can define a formula, F = F, where  is the coefficient for Rh123 fluorescence at 380 nm excitation. In our experiments,  was determined to 0.052, when average background fluorescence was subtracted. This can be used to subtract the Rh123 contribution from the total fluorescence at 380 nm excitation (F) in cells loaded with both fura-2 and Rh123. The true fura-2 signal at 380 nm excitation (F) was consequently calculated as F = F  F = F  F at every time point of the experiments.

Ability of Rh123 to Report Changes in Mitochondrial Membrane Potential at Different Loading Concentrations-- Low loading concentrations of Rh123 (2 µM = 0.8 µg/ml) gave rise to a substantial amount of fluorescence at 500 nm excitation, reaching levels 30 times above background fluorescence. However, -cells loaded with 2 µM Rh123 did not show a decrease in fluorescence, corresponding to hyperpolarization of the mitochondria, when exposed to 10 mM glucose (data not shown). Also, these cells displayed no increase in fluorescence, corresponding to depolarization, in response to 0.3, 1, or 3 mM azide (data not shown). Higher loading concentrations (8-32 µM) of Rh123 resulted in more intense fluorescence, and -cells loaded with these Rh123-concentrations also responded to stimulation with glucose and azide (data not shown). It seems that a threshold for mitochondrial uptake of Rh123 must be exceeded before the dye starts reporting changes in inner mitochondrial membrane potential.

Intracellular Distribution of Rh123 Measured with Laser Scanning Confocal Microscopy-- Rh123 has been used extensively as a fluorescent stain of mitochondria in living cells (8, 19, 20). As seen in Fig. 1 A, Rh123 was taken up by mitochondria-like intracellular structures in -cells. Fluorescence intensity, both from mitochondria-containing cytoplasmic areas and from mitochondria-free nuclear areas, increased with an increase in the loading concentration of Rh123 (Fig. 1B). Mitochondria were accumulated in the basal part of -cells close to the area of cellular attachment to the coverslip.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Rh123 accumulates in mitochondria and does not adversely affect cellular function. A, images showing Rh123 fluorescence in a single pancreatic -cell detected by confocal laser scanning microscopy. Two confocal sections of each cell are shown, one from the middle and one from the basal part. Cells were loaded with 3 µM fura-2/AM for 30 min and also with 2, 8, and 32 µM Rh123, respectively (n = 3 for each Rh123 concentration) for the last 10 min of incubation. The cells shown in this figure were loaded with 2 (cell 1) and 8 (cell 2) µM Rh123. B, histogram of Rh123 fluorescence in pancreatic -cells. Cells were incubated at different concentrations of Rh123, and fluorescence was measured by confocal microscopy. Filled bars represent nuclear areas, which can be considered mitochondria-free zones, and open bars cytosolic areas, including mitochondrial fluorescence (mean values ± S.E. for n = 4-16 in three different preparations of -cells). C, representative patch clamp experiment from a series of three showing that -cells previously exposed to 32 µM Rh123 for 10 min responded to stimulation with 10 mM glucose with depolarization and electrical activity.

Intriguingly, even a low loading concentration (2 µM) of Rh123 resulted in a substantial cellular uptake of the indicator, and the dye accumulated primarily in intracellular structures displaying the size and shape of mitochondria (Fig. 1A, cell 1). However, at this low loading concentration of Rh123, no responses to agents known to cause highly reproducible alterations of mitochondrial membrane potential were reported. Quenching of Rh123 fluorescence in mitochondria has been said to occur because of forced aggregation of dye molecules (8, 21). Depolarization of mitochondrial membrane potential results in dequenching of Rh123 fluorescence (9). Apparently, a minimum concentration of Rh123 molecules must be exceeded in the mitochondria for aggregation to occur.

Effect of Rh123 on Plasma Membrane Potential-- Rh123 has been demonstrated to inhibit mitochondrial respiration and partially purified mitochondrial F1-ATPase under certain conditions (19). If Rh123 exerts toxic effects on mitochondria in intact mouse -cells, ATP production is likely to become compromised. This would interfere with glucose stimulated electrical activity in the -cell. In order to investigate whether mitochondrial function was adversely affected by the dye, we exposed -cells to 32 µM Rh123 for 10 min. Using the perforated patch configuration of the patch clamp technique, it was demonstrated that glucose-stimulated electrical activity was unaffected by Rh123 (Fig. 1C).

Time Relationship between Onset of Mitochondrial Hyperpolarization and Change in [Ca²⁺]_i in Glucose-stimulated -Cells-- When -cells loaded with both an adequate concentration of Rh123 and fura-2 were stimulated with 10 mM glucose, the dyes reported hyperpolarization of mitochondrial membrane potential and changes in [Ca²⁺]_i (Fig. 2). For the most part, mitochondrial hyperpolarization preceded any change in [Ca²⁺]_i. In 10 experiments in a series of 22, mitochondrial hyperpolarization was followed by a decrease in [Ca²⁺]_i, the average lag phase being 6.5 ± 2.4 s. Whether or not a transient decrease in [Ca²⁺]_i occurred, mitochondrial hyperpolarization was usually followed by an increase in [Ca²⁺]_i. This increase in [Ca²⁺]_i followed 77 ± 6.8 s (n = 22, -cells from 9 mice) after the onset of hyperpolarization of the mitochondrial membrane potential. In glucose-stimulated insulin-producing cells, the initial change in [Ca²⁺]_i occurred on average 85 s after the initial change in oxygen consumption (22). It should be noted that in five of the experiments in the present study in which an initial decrease in [Ca²⁺]_i was observed, there was no lag time at all between mitochondrial hyperpolarization and the decrease in [Ca²⁺]_i.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Representative responses in mitochondrial membrane potential and [Ca2+]i to glucose stimulation. The addition of 10 mM glucose is indicated by the horizontal bar. The vertical dotted lines were included for easy comparison of traces at given time points. Hyperpolarization of mitochondrial membrane potential occurs simultaneously with or slightly before a decrease in [Ca2+]i. Later, an increase in [Ca2+]i is observed. The Rh123 contribution to fluorescence at 380 nm excitation was corrected for as described above. Both the scale representing Rh123 fluorescence and the scale representing fura-2 fluorescence have been inverted to give a more intuitive representation of [Ca2+]i and mitochondrial membrane polarization (n = 6).

It is also noteworthy that the rapid drop in emission at 500 nm excitation that occurred after glucose stimulation, indicating mitochondrial hyperpolarization, could obscure an initial rise of the simultaneously obtained raw emission signal at 380 nm excitation, corresponding to a decrease in [Ca²⁺]_i. This effect was due to contamination of the emitted signal at 380 nm excitation by Rh123, as noted, and was corrected for as described above. This correction thus revealed changes in [Ca²⁺]_i that would otherwise have escaped detection.

Effect of -Cell Stimulation on Mitochondrial Membrane Potential in the Absence of Extracellular Ca²⁺ and Effect of Plasma Membrane Depolarization on Rh123 Fluorescence-- The addition of 10 mM glucose to -cells in the absence of extracellular Ca²⁺ resulted in hyperpolarization of mitochondrial membrane potential without any effect on [Ca²⁺]_i (Fig. 3A). The time from onset of mitochondrial hyperpolarization until a new steady state level of membrane potential had been reached took 152 ± 17 s (n = 5, -cells from 3 mice). In the presence of extracellular Ca²⁺, this process took 62 ± 3 s (n = 5, -cells from 4 mice). The difference between the two groups was statistically significant (p < 0.001, Student's t test for unpaired data). The degree of glucose-induced hyperpolarization achieved, expressed as % reduction of fluorescence at 500 nm excitation, was 26 ± 2.1% in the absence of extracellular Ca²⁺ (n = 5, -cells from 3 mice). In the presence of normal extracellular Ca²⁺, the degree of hyperpolarization was 21.6 ± 2.8% (n = 5, -cells from 5 mice). The difference between glucose-induced hyperpolarization in the two groups did not reach statistical significance (Student's t test for unpaired data).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of glucose, tolbutamide, and KCl on mitochondrial membrane potential and [Ca2+]i in the absence of extracellular Ca2+. Mitochondrial membrane potential and [Ca2+]i were measured simultaneously. A, 10 mM glucose is added to a buffer from which Ca2+ was omitted. Cells exposed to low Ca2+ buffer from the start of the experiment. The normal extracellular Ca2+ concentration is reestablished later during the experiment. The addition of glucose and Ca2+ is indicated by horizontal bars. This is a representative experiment from a series of five performed on -cells from three different animals. B, the addition of 25 mM KCl and 100 µM tolbutamide to a buffer from which Ca2+ was omitted. Additions are indicated by horizontal bars. This is a representative experiment from a series of five performed on -cells from three different animals. Both the scale representing Rh123 fluorescence and the scale representing fura-2 fluorescence have been inverted to give a more intuitive representation of [Ca2+]i and mitochondrial membrane polarization. C, application of a depolarizing patch clamp pulse in a -cell loaded with Rh123. FCCP added to depolarize the inner mitochondrial membrane to prevent any subsequent contribution from the mitochondria to changes in the Rh123 signal. The addition of FCCP is indicated by the horizontal bar. No effect of the depolarizing pulse on the Rh123 signal is observed. This is a representative experiment from a series of six performed on -cells from three different animals.

The addition of tolbutamide or high K⁺ in the absence of extracellular Ca²⁺ had no effect on [Ca²⁺]_i but resulted in low amplitude changes in Rh123 fluorescence (Fig. 3B). To ensure that this was not due to a pool of Rh123 responding to changes in the plasma membrane potential, combined measurements of Rh123 fluorescence and plasma membrane potential using patch clamp were carried out (Fig. 3C). It was found that periods of depolarization at the plasma membrane had no effect on Rh123 fluorescence. These voltage clamp periods were given during conditions of mitochondrial membrane depolarization with FCCP to ensure that signaling of Rh123 was not affected by changes of polarization in the mitochondria.

Effect of Glucose Stimulation on NAD(P)H Fluorescence-- The addition of 10 mM glucose resulted in an increase in NAD(P)H fluorescence in a majority of cells (in at least 90% of 143 cells in 14 experiments). This corresponded with the number of cells displaying an increase in [Ca²⁺]_i under the same conditions (in at least 90% of 59 cells in 5 experiments). The increase in [Ca²⁺]_i was followed by significant oscillations (in at least 49% of the cells) with periods ranging from 184 to 360 s. Oscillations in NAD(P)H were difficult to detect because the low amplitude variations in NAD(P)H fluorescence were often of the same magnitude as the noise and instability in the excitation light (data not shown).

[Ca²⁺]_i and Mitochondrial Membrane Potential during Stimulation of -Cells-- Measurements of [Ca²⁺]_i simultaneously with mitochondrial membrane potential demonstrated that glucose-induced oscillations in [Ca²⁺]_i were not preceded by oscillations in mitochondrial membrane potential. On the contrary, brief depolarizations in mitochondrial membrane potential occurred after the [Ca²⁺]_i oscillations (Fig. 4A). Typically, after a [Ca²⁺]_i peak, a transient depolarization of mitochondrial membrane potential occurred. When [Ca²⁺]_i was forced to a stable, elevated level in glucose-stimulated cells previously shown to oscillate in both [Ca²⁺]_i and mitochondrial membrane potential, the latter oscillations also subsided (Fig. 4B). To stabilize [Ca²⁺]_i at an elevated level it was necessary to add a mixture of [Ca²⁺]_i-raising compounds consisting of 8.7 mM Ca²⁺, 100 µM tolbutamide, 25 mM KCl, and 1 µM BAY K8644.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Glucose-induced oscillations in [Ca2+]i. A, [Ca2+]i oscillations are followed by oscillations in mitochondrial membrane potential. Stimulation with 10 mM glucose resulted in oscillations in [Ca2+]i, which were followed by transient depolarizations in mitochondrial membrane potential. This pattern was observed in 7 of 10 experiments performed on -cells from at least three different animals. The Rh123 contribution to fluorescence at 380 nm excitation was corrected for as described above. Vertical dotted lines were included for easy comparison of traces at given time points. B, effect on mitochondrial membrane potential of stabilizing [Ca2+]i at an elevated level in a single -cell previously displaying glucose induced oscillations in [Ca2+]i and mitochondrial membrane potential. Oscillations in mitochondrial potential subside as [Ca2+]i stabilizes. The bar with the label "additions" refers to the addition of 8.7 mM Ca2+, 100 µM tolbutamide, 25 mM KCl, and 1 µM BAY K8644. Both the scale representing Rh123 fluorescence and the scale representing fura-2 fluorescence have been inverted to give a more intuitive representation of [Ca2+]i and mitochondrial membrane polarization. A moving average filter was applied using three neighboring data points. This is a representative experiment from a series of three.

Minor transient depolarizations of mitochondrial membrane potential also occurred after increases in [Ca²⁺]_i because of the addition of tolbutamide, KCl, and carbamylcholine to glucose-stimulated -cells (Fig. 5, A, B, and D). In contrast, tolbutamide-, KCl-, and carbamylcholine-induced increases in [Ca²⁺]_i at basal glucose caused a transient hyperpolarization of mitochondrial membrane potential (Fig. 5, A-C). Thus, a rise in [Ca²⁺]_i seems to affect mitochondrial membrane potential differently depending on the prevailing level of mitochondrial polarization.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Elevation of [Ca2+]i affects mitochondrial membrane potential differently depending on the prevailing level of mitochondrial polarization. The addition of tolbutamide (three of three experiments) (A), KCl (3 of 5 experiments) (B), or carbamylcholine (Cch) (four of four experiments) (D) to -cells already stimulated by 10-20 mM glucose first caused an increase in [Ca2+]i and then a transient depolarization in mitochondrial membrane potential. Tolbutamide (three of five experiments) (A), KCl (10 of 14 experiments) (B), or carbamylcholine (five of eight experiments) (C) added at basal glucose also resulted in an increase in [Ca2+]i, which was followed by transient hyperpolarization of the mitochondrial membrane potential. Vertical dotted lines were included for easy comparison of traces at given time points. Both the scale representing Rh123 fluorescence and the scale representing fura-2 fluorescence have been inverted to give a more intuitive representation of [Ca2+]i and mitochondrial membrane polarization.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study demonstrates that the typical response pattern in a glucose stimulated -cell is hyperpolarization of mitochondrial membrane potential, a simultaneous or somewhat delayed decrease in [Ca²⁺]_i, and thereafter an increase in [Ca²⁺]_i followed by a slight depolarization of mitochondrial membrane potential. When simultaneously registering [Ca²⁺]_i and mitochondrial membrane potential, we found that the time lag between onset of mitochondrial hyperpolarization and the increase in [Ca²⁺]_i after glucose stimulation agrees with similar data obtained by Duchen et al. (8) in experiments in -cells measuring the two parameters separately. Experiments performed when extracellular Ca²⁺ had been omitted show that mitochondrial hyperpolarization occurs upon glucose stimulation without any subsequent rise in [Ca²⁺]_i. However, the process of mitochondrial hyperpolarization was slower in the absence of extracellular Ca²⁺. Conceivably, activation of the Ca²⁺-sensitive mitochondrial dehydrogenases (23) occurs more rapidly during glucose stimulation in the presence of a physiological concentration of extracellular, and consequently intracellular, Ca²⁺ and thus speeds up the initial hyperpolarization of the inner mitochondrial membrane. When Ca²⁺ is omitted from the extracellular medium, depletion of intracellular Ca²⁺ will occur. Addition of high K⁺ or tolbutamide in the absence of extracellular Ca²⁺ caused a somewhat delayed transient elevation in the Rh123 signal. This seems not to be due to a pool of Rh123 molecules reporting depolarization at the level of the plasma membrane, as demonstrated by our experiments measuring Rh123 fluorescence in voltage clamped cells. Furthermore, Duchen (24) demonstrated that the Rh123 signal change in response to depolarization of mouse neurons with high K⁺ did not reflect plasma membrane depolarization. Our data suggest that, apparently, a stimulus that depolarizes the plasma membrane somehow signals to the mitochondria causing their inner membranes to transiently depolarize.

In several experiments, there was no lag phase at all between the onset of glucose-induced hyperpolarization of mitochondrial membrane potential and the initial decrease in [Ca²⁺]_i. Thus, there seems to be a direct correlation between increased energy supply by mitochondrial metabolism and lowering of the [Ca²⁺]_i. In a previous study of glucose-stimulated insulin-producing cells, the cellular ATP/ADP ratio increased 9 s before the initial increase in oxygen consumption (22), suggesting a close coupling between increased cellular energy charge and activation of mitochondrial respiration. In another study in which glucose-induced changes in [Ca²⁺]_i and plasma membrane potential in -cells were measured simultaneously, it was observed that the onset of the decrease in [Ca²⁺]_i occurred simultaneously with the onset of depolarization of plasma membrane potential (25). It was concluded that a nutrient-induced elevation of ATP leads to ATP-dependent removal of Ca²⁺ from the cytoplasm, paralleled by a slow depolarization of plasma membrane potential due to inhibition of ATP-sensitive K⁺ channels. Conversely, it is now clear that decreased ATP levels due to inhibition of mitochondrial function result in opening of K_ATP channels (26).

Increases in [Ca²⁺]_i caused by KCl or carbamylcholine stimulation resulted in different effects on mitochondrial membrane potential depending on whether the -cells had already been stimulated with glucose. An increase in [Ca²⁺]_i has complex effects on mitochondrial function. The mitochondrial free [Ca²⁺] is elevated (27, 28), and the influx of [Ca²⁺] into the mitochondria results in net depolarization of their membrane potential (24, 29, 30). Also, mitochondrial dehydrogenases are activated (23), and this tends to hyperpolarize the mitochondria. The net effect of a [Ca²⁺]_i-peak on mitochondrial membrane potential could thus depend on the conditions prevailing at the time, e.g. the ambient glucose concentration.

The main focus of the present study was to measure mitochondrial membrane potential simultaneously with [Ca²⁺]_i oscillations in glucose-stimulated -cells. We found that glucose-induced [Ca²⁺]_i-oscillations in pancreatic -cells precede oscillations in mitochondrial membrane potential. Peaks in [Ca²⁺]_i are followed by rapid depolarizations of mitochondrial membrane potential. These mitochondrial depolarizations are of similar amplitude and duration as those seen after high K⁺ or tolbutamide in the absence of extracellular Ca²⁺. Possibly, any stimulus that depolarizes the plasma membrane will also cause transient depolarization of the polarized mitochondrial inner membrane. It is not clear what signaling mechanism could mediate this effect. However, an influence from peaks in [Ca²⁺]_i on mitochondrial potential cannot be excluded in the case of glucose stimulation in the presence of extracellular Ca²⁺. When [Ca²⁺]_i was forced to a stable, elevated level in glucose-stimulated cells previously shown to oscillate in both [Ca²⁺]_i and mitochondrial membrane potential, the latter oscillations also subsided. A transient depolarization of the mitochondria was observed following the initial peak in [Ca²⁺]_i during glucose stimulation irrespective of whether regular oscillations in [Ca²⁺]_i occurred subsequently. That the initial transient depolarization of the mitochondria was due to influx of Ca²⁺ is supported by the fact that this effect was not observed in -cells stimulated by glucose in the absence of extracellular Ca²⁺. A pacing effect of pulsatile [Ca²⁺]_i changes on -cell metabolism has been demonstrated previously (31).

Our findings are compatible with a model of regulation of -cell [Ca²⁺]_i oscillations in which [Ca²⁺]_i-nadirs occur because of temporary inhibition of mitochondrial metabolism caused by the previously elevated [Ca²⁺]_i. This feed-back loop was recently suggested also by Krippeit-Drews et al. (11). The model is compatible with observations in single mouse islets that oscillations in oxygen consumption are dependent on the presence of extracellular Ca²⁺ or influx of Ca²⁺ through voltage-gated Ca²⁺ channels (32). However, in a recent report demonstrating oscillations in oxygen consumption in glucose-stimulated single clonal -cells in the absence of extracellular Ca²⁺, it was argued that Ca²⁺ cannot be the primary generator for oscillations in oxygen consumption (33). The presence of oscillations in oxygen consumption per se in glucose-stimulated pancreatic islets is however clearly established (34, 35).

Another possible explanation for the presence of glucose-induced [Ca²⁺]_i oscillations is that these occur because of fluctuations in the cytoplasmic ATP/ADP ratio due to oscillations in glycolysis. Evidence suggesting glycolytic oscillations in glucose-stimulated rat islets (36) and in synchronized suspensions of mouse -cells (37) has been presented. Oscillations in -cell glycolysis occurring as a consequence of the properties of the glycolytic enzyme phosphofructokinase have been suggested to cause oscillations in K_ATP channels and [Ca²⁺]_i (38). In addition, local events close to the ion channels in the plasma membrane, e.g. local ATP consumption at the sites of plasma membrane Ca²⁺-pumps, could play a role in the regulation of glucose-induced [Ca²⁺]_i oscillations. Such local events could regulate the activity of plasma membrane K_ATP channels.

Transient inhibition of cellular metabolism is expected to inhibit the reduction of NAD(P) to NAD(P)H. We measured cellular NAD(P)H levels as reflected by autofluorescence levels and, using spectral analysis, tried to detect low amplitude oscillations in NAD(P)H fluorescence Unfortunately, the amplitude and period of fluctuations in NAD(P)H fluorescence were often similar to those of spontaneously and sporadically occurring oscillations in excitation light intensity. Therefore, we were unable to demonstrate unequivocally the presence of oscillations in NAD(P)H.

The present study evaluates the method of simultaneous measurements of mitochondrial membrane potential and [Ca²⁺]_i in the pancreatic -cell using fluorescent dyes. We have clearly shown that glucose-induced [Ca²⁺]_i peaks in these cells are followed by mitochondrial membrane depolarization episodes. These mitochondrial depolarizations may coincide with episodes of decreased mitochondrial ATP production and thus result in the transient opening of plasma membrane K_ATP channels, plasma membrane repolarization, temporary closure of L-type Ca²⁺ channels, and transient lowering of the [Ca²⁺]_i. We have also demonstrated that the effect on mitochondrial membrane potential of agents that depolarize the plasma membrane, or that release Ca²⁺ from intracellular stores, is dependent on ambient glucose concentration and/or the extracellular Ca²⁺ level.

	FOOTNOTES

^* Financial support was obtained from the Swedish Medical Research Council (Grants 03X-09890, 03XS-12708, 03X-09891, and 19X-00034), the Swedish Diabetes Association, the Juvenile Diabetes Foundation International, the Nordic Insulin Foundation, the Novo Nordisk Foundation, Funds of Karolinska Institutet, Clas Groschinskys Memorial Foundation, Magnus Bergvall's Foundation, funds of the Swedish Society of Medicine, Fredrik och Ingrid Thurings Stiftelse, United States Public Health Service Grant DK-35 914, Berth von Kantzows Foundation, and Tore Nilsson's Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this study.

^§ To whom correspondence should be addressed. Tel.: 46-8-5177-5731; Fax: 46-8-517-79450; E-mail: henrik.kindmark@ks.se.

Published, JBC Papers in Press, July 9, 2001, DOI 10.1074/jbc.M102492200

	ABBREVIATIONS

The abbreviations used are: K_ATP channels, ATP-dependent K⁺ channels; [Ca²⁺]_i, cytoplasmic free calcium concentration; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; fura-2/AM, fura-2/acetoxymethylester; Rh123, rhodamine 123; cps, counts per second.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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